We have been developing strategies to utilize nitric oxide (NO) in cancer treatment. In addition to the use of synthetic NO donors, we are researching the mechanisms that control the endogenous cellular production. We have recently discovered that inhibition of Nitric oxide synthase (NOS) given after chemotherapy or radiation treatment enhances tumor re-growth delay. Given that there are a number of clinically available NOS inhibitors this could have potential clinical applications. We have recently characterized the NO levels that are required to activate and stabilize key proteins involved in carcinogenesis, p53, ERK and HIF1-alpha. We have extended this work to show that levels of NO are critical to angiogenesis and that NO and TSP-1 regulate each other pathways. Our extension of the understanding of angiogenic responses and nitric oxide has lead us to understand the regulation of Matrix Metalloproteases (MMP) by NO . Our findings suggest that under inflammatory response MMP are regulated by two different mechanisms, biologocal signlaing and direct chemical modification. The first is via TIMP through cGMP. The second are through chemical reactivity of RNS. We demonstrated that MMP9 secreted from macrophage was a critical factor in the NO mediated wound healing response. Our recent discover that NOS-2 and COX-2 are strong predictor of poor prognosis in ER negative cancer. In patient cohort we have found that 94% of the deseased had high NOS2 in ER- patients. We have been exploring the molecular mechanisms that elucidate pathways that link these biomarkers. We have found that NO has two effect: one it activates key pathways, beta-catenin, NFkappa, AP-1 and ERK. These pathways lead to increase proliferation, migration and cancer stem cell markers. These findings have provided new models to develop compounds that target this particular aggressive cancer type describe in project 3 and 4. One of the unique aspect is that NO can induce many markers that associated with poor prognosis in breast cancer. However, activation of the pathways are not enough, there has to be inhibition of tumor supressor proteins. We have found that BRCA1 and PP2A are both inhibited by NO. This molecular profile points to a unique mechanism that suggest NO and NOS-2 are complete protumorigenic agent with implications in breast, colon, liver, brain, prostate, stomach and pancreatic cancer.